EP1804822B1 - Hcv vaccines for chronic hcv patients - Google Patents
Hcv vaccines for chronic hcv patients Download PDFInfo
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- EP1804822B1 EP1804822B1 EP05794493A EP05794493A EP1804822B1 EP 1804822 B1 EP1804822 B1 EP 1804822B1 EP 05794493 A EP05794493 A EP 05794493A EP 05794493 A EP05794493 A EP 05794493A EP 1804822 B1 EP1804822 B1 EP 1804822B1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- HCV Hepatitis C Virus
- HCV is a major cause of cirrhosis, end-stage liver disease and liver cancer (Liang 2000).
- Strength and quality of both HTL and CTL responses determine whether patients recover (spontaneously or as a consequence of therapy) or develop chronic infection (Liang 2000).
- Standard therapy of HCV comprises a combination of pegylated interferon-alpha and the antiviral ribavirin (Fried 2002).
- Virologic responses are, depending on the genotype, however, achieved in only about 50% of HCV patients with the standard therapy.
- IFN interferon
- ribavirin a compound that stimulates the immune system in a non-specific manner, which causes substantival side effects including flu-like syndrome, fever, headache, arthralgia, myalgia, depression, weight loss, alopecia, leukopenia and thrombopenia 6 .
- the WO 01/81359 A1 discloses ribavirin derivatives for the treatment of chronic hepatitis C infections.
- the problem underlying the present invention is to overcome the limitations of the standard interferon-alpha/ribavirin combination therapy by providing effective medicaments or pharmaceutical compositions especially a vaccine for the treatment of chronic HCV infections, especially for those patients who had not responded to or only partially responded to or had relapsed from primary standard HCV therapy.
- the present invention provides the use of polycationic peptides for the preparation and manufacturing of medicaments or pharmaceutical compositions, comprising HCV antigens or HCV epitopes for the treatment of patients with HCV chronic infections.
- the patients with HCV chronic infections are those who had not responded to, partially responded to or had relapsed from primary standard HCV therapy by a combination of pegylated interferon-alpha and the antiviral ribavirin.
- the medicaments or pharmaceutical compositions are preferably vaccines.
- a patient is considered to have relapsed when HCV RNA becomes undetectable on the primary standard HCV therapy with Peg-interferon alpha and Ribavirin but is detected again after disclontinuation of the treatment.
- Persons in whom HCV RNA levels remain stable on treatment are considered non-responders, while those whose HCV RNA levels decline (e.g. by >2 logs), but never become undetectable, are referred to as partial responders.
- the polycationic peptide(s) to be used according to the present invention may be any polycationic peptide, which shows the characteristic effects according to the WO 97/30721 .
- Preferred polycationic peptides are selected from basic polyppetides, organic polycations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues ( WO 97/30721 ).
- polypeptides e.g. polyethyleneimine
- WO 99/38528 e.g. polyethyleneimine
- these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.
- polycationic peptides may be produced chemically or recombinantly or may be derived from natural sources.
- Cationic (poly)peptides may also be anti-microbial with properties as reviewed in ⁇ Ganz, T., 1999 ⁇ . These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly ( WO 02/13857 ). Peptides may also belong to the class of defensins ( WO 02/13857 ). Sequences of such peptides can be, for example, found in the Antimicrobial Sequences Database under the following internet address: http://www.bbcm.univ.trieste.it/ ⁇ tossi/pag2.html
- Such host defence peptides or defensives are also a preferred form of the polycationic polymer according to the present invention.
- a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.
- polycationic peptides in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (International patent application WO 02/13857 ), especially antimicrobial peptides derived from mammalian cathelicidin, preferably from human, bovine or mouse.
- Polycationic peptides derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production.
- Other preferred polycationic peptides are cathelin or related or derived substances from cathelin.
- mouse cathelin is a peptide, which has the amino acid sequence NH 2 -RLAGLLRKGGEKIGEKLKKIGQKIKNEFQKLVPQPE-COOH.
- Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues.
- Derivations may include the substitution or modification of the natural amino acids by amino adds, which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the antigen. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as evident adjuvants in vaccine formulations with or without further immunactivating substances.
- Another preferred polycationic peptide to be used according to the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (International patent application WO 02/32451 .
- a type 1 inducing adjuvant (Immunizer) that is able to strongly enhance the immune response to a specific co-administered antigen and therefore constitutes a highly effective adjuvant is disclosed.
- the adjuvant (Immunizer) is a peptide comprising a sequence R1-XZXZNXZX-R2, whereby N is a whole number between 3 and 7, preferably 5, X is a positively charged natural and/or non-natural amino acid residue, Z is an amino acid residue selected from the group consisting of L, V, I, F and/or W, and R1 and R2 are selected independantly one from the other from the group consisting of -H, -NH2, -COCH3, -COH, a peptide with up to 20 amino acid residues or a peptide reactive group or a peptide linker with or without a peptide; X-R2 may also be an amide, ester or thioester of the C-terminal amino acid residue.
- a specifically preferred peptide is KLKLLLLLKLK.
- the polycationic peptides can be used for the manufacturing of any medicaments, pharmaceutical compositions, especially vaccines which can be used for the treatment of chronic HCV infection, particularly for the treatment of those patients who had not responded to, partially responded to or had relapsed from primary standard HCV therapy by a combination of pegylated interferon-alpha and the antiviral ribavirin.
- the polycationic peptides are used for the manufacturing of medicaments, pharmaceutical compositions, especially vaccines that comprise HCV antigens, HCV hotspot epitopes and HCV epitopes.
- vaccines that comprise HCV antigens, HCV hotspot epitopes and HCV epitopes.
- HCV epitopes that are described in WO 01/24822 and PCT/EP2004/007540 .
- HCV epitopes therefore include: one or more, especially two, three, four, five or six of the following epitopes: MWNFISGIQYLAGLSTLPGN, HMWNHSGI, NHSGIQYLAGLSTLPGNPA, HMWNFISGIQYLAGLSTLPGNPA, IGLGKVLVDILAGYGAGVAGALVAFK AAWYELTPAETTVRLR, DYPYRLWHYPCTVNYTIFKI, DYPYRLWHYPCTVNFTIFKI, AYSQQTRGLL, TAYSQTRGLLG, SMSYTWTGALITP, SPGALWGVI, LPRRGPRL, IGLGKVLVDILAGYgAGVAGALVAFK, GSIGLGKVLVDILAG, IGLGKVLVDILAGYG, LGKVLVDILAGYGAG, LVDILAGYGAGVAGA, DILAGYGAGVAGALV, LAGYGAGVAGALVAF, AAW
- the vaccine according to the present invention contains two or more, even more preferred three or more, especially four or more of such epitopes in combination.
- Preferred vaccines contain 3, 4, 5, 6 or 7 individual epitopes in one preparation (or two preparations stored and reconstituted seperately and applied together).
- the medicament used according to the present invention is preferably used for induction of CD4+ Helper-T-cells and CD8+ cytotoxic T-cells.
- a specifically preferred field of use is the application of the medicament to a special group of patients: the use of the present medicament for replacing or supplementing a HCV standard therapy with interferon alpha and ribavirin, especially in patients where such standard therapy is not effective or not effective anymore.
- the medicament according to the present invention is used in combination with standard treatment such as interferon treatment.
- standard treatment such as interferon treatment.
- it can be used for inducing type I T-cell responses in chronic HCV patients, and/or for inducing similar T-cell responses as seen during/after successful standard therapy, and/or for increasing responder rates and/or reducing relapse rates after standard therapy.
- the medicament according to the present invention is used in clinical trials as late add-on to standard therapy; the results confirm the excellent safety of the medicament, which does not exacerbate the side-effects of standard therapy such as leukopenia.
- the medicament according to the present invention is specifically designed for HCV genotype 1. Genotype 1 patients have shown the lowest responder rates during standard therapy. Thus due to its ability to induce type I T-cell responses in chronic HCV patients, the medicament according to the present invention is preferably used to replace or supplement ribavirin (i.e. IFN/IC41 instead IFN/riba).
- ribavirin i.e. IFN/IC41 instead IFN/riba
- the medicament according to the present invention can be also used in combination with small-molecule protease inhibitors. Preferably, it can be used for inducing type I T-cell responses in chronic HCV patients and/or for inducing similar T-cell responses as seen during/after successful standard therapy.
- the medicament according to the present invention hence can offer a mode-of-action distinctly different from small-molecule inhibitors. It can hence complement efficacy of small molecule inhibitors in terms of response rates, duration of response, relapse rates, optimal dose/schedule.
- the medicament according to the present invention has no significant side effects, can in particular improve the efficacy/side-effect ratio of a small molecule inhibitor.
- the medicament according to the present invention can be used in combination with imiquimod (a TLR7 agonist in the already licensed product Aldara/3M).
- imiquimod a TLR7 agonist in the already licensed product Aldara/3M.
- the local mobilization of antigen-presenting cells can increase the immunological potency of the medicament according to the present invention.
- stronger responses against more peptides after less vaccinations can be expected; the immunological responder rates and duration of responses and hence also virological responses can be increased; the stronger T-cell responses against more peptides can overcome the RNA rebound effect e.g. as seen in the IC41-201 study using the medicament according to the present invention.
- the present invention also encompasses a method of treating the HCV patients described herein with an effective amount of the described medicament.
- Example I Human clinical study of HCV peptide vaccine in chronic HCV patients:
- the first vaccine where Poly-L-Arginine has been applied in humans is a fully synthetic therapeutic Hepatitis C Virus (HCV) vaccine.
- HCV Hepatitis C Virus
- This vaccine was named IC41 and consists of a mixture of synthetic peptides representing conserved T cell epitopes of HCV plus Poly-L-Arginine as a synthetic T cell adjuvant.
- IC 41 comprises five peptides from different regions from the HCV polypeptide, i.a. the following three epitopes: HMWNFISGIQYLAGLSTLPGNPA, CINGVCWTV and DLMGYIPAV.
- the aim of this therapeutic approach is to restore a so-called type I T cell response against HCV in chronically infected patients.
- T cell stimulatory efficacy of Poly-L-Arginine was tested in a phase 2 clinical trial in chronic Hepatitis C Virus patients, who did not respond to or relapsed from standard interferon/ribavirin therapy.
- HCV peptide vaccine IC41
- IC41 chronic HCV who had not responded to or had relapsed from primary standard HCV therapy.
- the study was conducted in 11 centers in Germany, Austria and Trunty patients were randomly assigned to receive three different doses and rations of HCV peptide vaccine with Poly-L-Arginine, HCV peptide vaccine alone or Poly-L-Arginine alone.
- each group consisted of 12 subjects, positive for HLA-A2. Subjects received 6 vaccinations in monthly intervals (at visits 3 to 8). Blood for immunological analyses was drawn prior vaccination and at visits 6 to 8, one month after last vaccination (visit 9), 3 months after last vaccination (visit 10) and 6 months after last vaccination (visit 11).
- state-of-the-art T cell assays to determine immunological endpoints under GLP/GCP compliance were applied: Interferon-gamma ELIspot Assay, T cell Proliferation Assay, HLA-tetramer/FACS assay. These assays allow reliable measurements of epitope-specific T cell responses induced by the therapeutic HCV vaccine IC41. The vaccine-induced T cell immune responses serve as surrogate parameters of efficacy (Keilholz et al. 2002).
- T-cell proliferation assay allows detection of peptide-specific T cells in biological samples like human blood.
- the basis of the assay is that, T cells upon stimulation with a peptide specifically recognized by their T cell receptor, react by secretion of cytokines and subsequent proliferation. Proliferation of cells can be measured by a variety of means; among the most sensitive approaches ranks incorporation of radioactively labeled thymidine into DNA synthesized prior cell division. This reaction can be carried out in a 96-well plate. Each well contains a fixed number of cells, which are cultured in the presence of antigen/peptide for a couple of days.
- thymidine labelled with tritium 3H-thymidine
- 3H-thymidine 3H-thymidine
- Cells are then harvested onto a filter plate: medium containing free radioactivity is washed away, whereas DNA sticks to the filter.
- Incorporated radioactivity can be quantified by means of a beta-scintillation counter determining counts-per-minute (cpm).
- the usual output is given as stimulation index (S.I.), which is defined as cpm of the test sample divided through cpm of the negative control.
- T-cell immunogenicity was determined by interferon gamma ELIspot.
- ELIspot allows quantification of peptide-specific, functional (i.e. cytokine- secreting) T cells in biological samples like human blood.
- the basis of the assay is that, T cells upon stimulation with a peptide specifically recognized by the T cell receptor react by secretion of cytokines like IFN- ⁇ . This reaction can be carried out in a 96-well plate. The filter-wells of this plate are coated with a Mab specific for IFN- ⁇ . Consequently, each cell secreting IFN- ⁇ leaves an IFN- ⁇ spot, which can be visualized with a subsequent color reaction. Spots can be counted using automated plate readers. Numbers obtained are a measure for the frequency of peptide-specific, IFN- ⁇ -secreting T cells in the sample.
- HLA class I tetramers soluble recombinant forms of a complex of HLA molecule and antigenic peptide, bind the antigen-specific T cell receptor used for T cell recognition.
- flow cytometry with fluorescent tetramers antigen-specific CD8 + T lymphocytes can be reliably enumerated and characterized.
- the assay uses HLA-A*0201 custom-made iTagTM-tetramers produced by Beckman Coulter Immunomics complexed with IC41 class I epitopes.
- Subjects were classified as responders if they showed significant T-cell responses at any of visits 4 to 11 and had no response prior treatment.
- pre-existing immunity significant T-cell response against any peptide within IC41 already prior vaccination
- an increase of at least 3 times of the pre-existing value was required to classify the effect as response.
- T cell immunity against the virus can be raised to a level that is not too different from the one induced in healthy vaccines. Thus, immunosuppression may not be as prevalent as anticipated in patients. It remains to be elucidated, how such T cell responses can be optimally applied to reduce disease progression or ameliorate symptoms and eventually clear the infection.
- Poly-L-Arginine represents one of the first synthetic T cell adjuvants, which has consistently - from in vitro experiments up to incurable chronically infected patients - been able to induce and augment the desired kind of immune response. Its easy manufacturability, excellent safety profile and its efficacy even in such difficult settings as chronic HCV infection, make it a promising new tool in the fight against infectious diseases and cancer.
- Example II CD4+ Helper-T-cell proliferation in chronic HCV patients can be induced by IC41, but not by peptides alone, or poly-L-Arginine alone
- IC41 is able of inducing a significant proliferative response in chronic HCV patients.
- neither peptides alone nor poly-L-Arginine alone have this ability, proofing the adjuvant effect of poly-L-Arginine.
- Example III poly-L-Arginine is required to induce type I (interferon gamma) T-cell responses in chronic HCV patients
- IC41 can induce interferon gamma secreting T-cells in chronic HCV patients, whereas peptides alone or poly-L-Arginine alone cannot induce any response.
- peptides alone or poly-L-Arginine alone cannot induce any response.
- both CD4+ Helper-T-cells and CD8+ cytotoxic T-cells can be induced.
- Interferon gamma secretion is a hallmark of type I T-cell responses. Such responses are seen during the acute phase of infection in the subset of HCV patients, who eliminate the virus and do not proceed to chronic infection. Type I T-cell responses are also seen in patients undergoing standard therapy with interferon and ribavirin. Thus, induction of type I T-cell responses as achieved by IC41 is a primary goal of therapeutic vaccination against HCV.
Description
- Hepatitis C Virus (HCV) is a member of the flaviviridiae chronically infecting about 170 million people worldwide. There are at least 6 HCV genotypes and more than 50 subtypes have been described. In America, Europe and Japan
genotypes - Standard therapy of HCV comprises a combination of pegylated interferon-alpha and the antiviral ribavirin (Fried 2002). Virologic responses (defined as the absence of detectable HCV RNA 24 weeks after cessation of therapy) are, depending on the genotype, however, achieved in only about 50% of HCV patients with the standard therapy. Moreover, there are many side effects associated with both interferon (IFN) and ribavirin. IFN (also in its pegylated form) stimulates the immune system in a non-specific manner, which causes substantival side effects including flu-like syndrome, fever, headache, arthralgia, myalgia, depression, weight loss, alopecia, leukopenia and thrombopenia6. These side effects can necessitate cessation of treatment for some subjects. Treatment with IFN is also contraindicated in subjects with pre-existing hematologic disease (leukopenia and thrombopenia due to liver cirrhosis with splenomegaly). Ribavirin can cause transient liver enzyme elevations and psychiatric symptoms, hemolysis and anemia and has been associated with myocardial infarction in subjects with coronary heart disease. Ribavirin also exhibits teratogenic, mutagenic and carcinogenic potency. Therefore, contraception is mandatory for fertile male and female subjects treated with ribavirin. Therefore, the low tolerability and the considerable side effects of this therapy clearly necessitate novel therapeutic intervention including therapeutic vaccines and new treatment options for chronic HCV infection are urgently required, due to the limitations and lack of effective treatments (Cornberg 2002).
- The
WO 01/81359 A1 - The problem underlying the present invention is to overcome the limitations of the standard interferon-alpha/ribavirin combination therapy by providing effective medicaments or pharmaceutical compositions especially a vaccine for the treatment of chronic HCV infections, especially for those patients who had not responded to or only partially responded to or had relapsed from primary standard HCV therapy.
- Therefore the present invention provides the use of polycationic peptides for the preparation and manufacturing of medicaments or pharmaceutical compositions, comprising HCV antigens or HCV epitopes for the treatment of patients with HCV chronic infections. Preferably, the patients with HCV chronic infections are those who had not responded to, partially responded to or had relapsed from primary standard HCV therapy by a combination of pegylated interferon-alpha and the antiviral ribavirin. The medicaments or pharmaceutical compositions are preferably vaccines.
- A patient is considered to have relapsed when HCV RNA becomes undetectable on the primary standard HCV therapy with Peg-interferon alpha and Ribavirin but is detected again after disclontinuation of the treatment. Persons in whom HCV RNA levels remain stable on treatment are considered non-responders, while those whose HCV RNA levels decline (e.g. by >2 logs), but never become undetectable, are referred to as partial responders.
- The polycationic peptide(s) to be used according to the present invention may be any polycationic peptide, which shows the characteristic effects according to the
WO 97/30721 WO 97/30721 WO 97/30721 WO 99/38528 - These polycationic peptides may be produced chemically or recombinantly or may be derived from natural sources.
- Cationic (poly)peptides may also be anti-microbial with properties as reviewed in {Ganz, T., 1999}. These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly (
WO 02/13857 WO 02/13857
http://www.bbcm.univ.trieste.it/∼tossi/pag2.html - Such host defence peptides or defensives are also a preferred form of the polycationic polymer according to the present invention. Generally, a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.
- Especially preferred for use as polycationic peptides in the present invention are cathelicidin derived antimicrobial peptides or derivatives thereof (International patent application
WO 02/13857 - Polycationic peptides derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production. Other preferred polycationic peptides are cathelin or related or derived substances from cathelin. For example, mouse cathelin is a peptide, which has the amino acid sequence NH2-RLAGLLRKGGEKIGEKLKKIGQKIKNEFQKLVPQPE-COOH. Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues. Derivations may include the substitution or modification of the natural amino acids by amino adds, which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the antigen. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as evident adjuvants in vaccine formulations with or without further immunactivating substances.
- Another preferred polycationic peptide to be used according to the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids (International patent application
WO 02/32451 WO 02/32451 type 1 inducing adjuvant (Immunizer) that is able to strongly enhance the immune response to a specific co-administered antigen and therefore constitutes a highly effective adjuvant is disclosed. The adjuvant (Immunizer) according to theWO 02/32451 - According to the present invention, the polycationic peptides can be used for the manufacturing of any medicaments, pharmaceutical compositions, especially vaccines which can be used for the treatment of chronic HCV infection, particularly for the treatment of those patients who had not responded to, partially responded to or had relapsed from primary standard HCV therapy by a combination of pegylated interferon-alpha and the antiviral ribavirin.
- Preferably, the polycationic peptides are used for the manufacturing of medicaments, pharmaceutical compositions, especially vaccines that comprise HCV antigens, HCV hotspot epitopes and HCV epitopes. Most preferably, the HCV epitopes that are described in
WO 01/24822 PCT/EP2004/007540 - The medicament used according to the present invention is preferably used for induction of CD4+ Helper-T-cells and CD8+ cytotoxic T-cells. A specifically preferred field of use is the application of the medicament to a special group of patients: the use of the present medicament for replacing or supplementing a HCV standard therapy with interferon alpha and ribavirin, especially in patients where such standard therapy is not effective or not effective anymore.
- Preferably, the medicament according to the present invention is used in combination with standard treatment such as interferon treatment. Preferably, it can be used for inducing type I T-cell responses in chronic HCV patients, and/or for inducing similar T-cell responses as seen during/after successful standard therapy, and/or for increasing responder rates and/or reducing relapse rates after standard therapy. In particular, the medicament according to the present invention is used in clinical trials as late add-on to standard therapy; the results confirm the excellent safety of the medicament, which does not exacerbate the side-effects of standard therapy such as leukopenia.
- Preferably the medicament according to the present invention is specifically designed for
HCV genotype 1.Genotype 1 patients have shown the lowest responder rates during standard therapy. Thus due to its ability to induce type I T-cell responses in chronic HCV patients, the medicament according to the present invention is preferably used to replace or supplement ribavirin (i.e. IFN/IC41 instead IFN/riba). - The medicament according to the present invention can be also used in combination with small-molecule protease inhibitors. Preferably, it can be used for inducing type I T-cell responses in chronic HCV patients and/or for inducing similar T-cell responses as seen during/after successful standard therapy. The medicament according to the present invention hence can offer a mode-of-action distinctly different from small-molecule inhibitors. It can hence complement efficacy of small molecule inhibitors in terms of response rates, duration of response, relapse rates, optimal dose/schedule. The medicament according to the present invention has no significant side effects, can in particular improve the efficacy/side-effect ratio of a small molecule inhibitor.
- The medicament according to the present invention can be used in combination with imiquimod (a TLR7 agonist in the already licensed product Aldara/3M). Especially the local mobilization of antigen-presenting cells can increase the immunological potency of the medicament according to the present invention. In particular, stronger responses against more peptides after less vaccinations can be expected; the immunological responder rates and duration of responses and hence also virological responses can be increased; the stronger T-cell responses against more peptides can overcome the RNA rebound effect e.g. as seen in the IC41-201 study using the medicament according to the present invention.
- The present invention also encompasses a method of treating the HCV patients described herein with an effective amount of the described medicament.
- The present invention is further illustrated by the following figures, examples from which further features, embodiments and advantages may be taken. It is to be understood that the present examples are given by way of illustration only and not by way of limitation of the disclosure.
-
Figure 1 : CD4+ Helper-T-cell proliferation in chronic HCV patients. IC41 but not peptides alone, or poly-L-Arginine alone can induce significant proliferation. Vaccinations were given atmonth -
Figure 2 : Interferon-gamma ELIspot Responder Rates of patients vaccinated with IC41 or peptide alone or poly-L-Arginine alone. Each group consisted of 12 patients. Top panel: CD4+ Helper- and CD8+ cytotoxic T-cells, bottom panel: CD8+ cytotoxic T-cell, only. - The first vaccine, where Poly-L-Arginine has been applied in humans is a fully synthetic therapeutic Hepatitis C Virus (HCV) vaccine. This vaccine was named IC41 and consists of a mixture of synthetic peptides representing conserved T cell epitopes of HCV plus Poly-L-Arginine as a synthetic T cell adjuvant. IC 41 comprises five peptides from different regions from the HCV polypeptide, i.a. the following three epitopes: HMWNFISGIQYLAGLSTLPGNPA, CINGVCWTV and DLMGYIPAV. The aim of this therapeutic approach is to restore a so-called type I T cell response against HCV in chronically infected patients. Such a response is typically seen in the around 15% of infected persons who do not proceed to chronicity but can clear HCV during the acute phase of infection. Since the pre-clinical experience with Poly-L-Arginine described earlier has shown its ability to induce type I immune responses in animal models it represents a promising T cell adjuvant for peptide vaccines for the treatment of HCV.
- Various doses of the mentioned vaccine have already been tested in several clinical trials comprising more than 200 subjects: in an
initial phase 1 study, safety and preliminary immunogenicity data of several doses were obtained. Results from that trial prompted initiation of a dose optimization study comprising 128 healthy volunteers in 10 different dose groups. The study was a randomized, single blind, parallel group, controlled study conducted to assess dose optimization and safety of the HCV peptide vaccine, IC41, in healthy subjects and was conducted in one center in Austria. One-hundred and twenty-eight subjects were randomly assigned to receive one of seven different doses and ratios of HCV peptide vaccine with Poly-L-Arginine, HCV peptide vaccine alone, Poly-L-Arginine alone or saline solution. All subjects received four administered vaccinations in monthly intervals. Immunogenicity was assessed at each of these time points at one month respectively and three months after the last vaccination. - The T cell stimulatory efficacy of Poly-L-Arginine was tested in a
phase 2 clinical trial in chronic Hepatitis C Virus patients, who did not respond to or relapsed from standard interferon/ribavirin therapy. - For the present invention, a randomized, double blind study of HCV peptide vaccine, IC41, was conducted in patients with chronic HCV who had not responded to or had relapsed from primary standard HCV therapy. The study was conducted in 11 centers in Germany, Austria and Poland. Sixty patients were randomly assigned to receive three different doses and rations of HCV peptide vaccine with Poly-L-Arginine, HCV peptide vaccine alone or Poly-L-Arginine alone.
- Male and female patients who met the following inclusion criteria were included in the study:
- Diagnosis of chronic hepatitis C, with a documented course of at least six months (ALT > 1.5 times the upper limit of normal [ULN] at two or more timepoints).
- Non-response to or relapse from primary standard HCV therapy of six to 12 months (depending on the genotype of HCV).
- HCV-RNA positive.
- HLA-A2 positive.
- HCV antibodies positive.
- Liver biopsy within 30 months prior to inclusion, demonstrating hepatic inflammation and/or fibrosis.
- Hematology and biochemistry laboratory results within the limits normally expected for the patient population (liver values maximum of 5x ULN).
- Aged 18 to 65 years.
- Written informed consent obtained prior to study entry.
- In the present clinical study, each group consisted of 12 subjects, positive for HLA-A2. Subjects received 6 vaccinations in monthly intervals (at
visits 3 to 8). Blood for immunological analyses was drawn prior vaccination and atvisits 6 to 8, one month after last vaccination (visit 9), 3 months after last vaccination (visit 10) and 6 months after last vaccination (visit 11). For immunological monitoring of clinical trials, state-of-the-art T cell assays to determine immunological endpoints under GLP/GCP compliance were applied: Interferon-gamma ELIspot Assay, T cell Proliferation Assay, HLA-tetramer/FACS assay. These assays allow reliable measurements of epitope-specific T cell responses induced by the therapeutic HCV vaccine IC41. The vaccine-induced T cell immune responses serve as surrogate parameters of efficacy (Keilholz et al. 2002). - As primary endpoint T-cell immunogenicity was determined by a T-cell proliferation assay. The proliferation assay allows detection of peptide-specific T cells in biological samples like human blood. The basis of the assay is that, T cells upon stimulation with a peptide specifically recognized by their T cell receptor, react by secretion of cytokines and subsequent proliferation. Proliferation of cells can be measured by a variety of means; among the most sensitive approaches ranks incorporation of radioactively labeled thymidine into DNA synthesized prior cell division. This reaction can be carried out in a 96-well plate. Each well contains a fixed number of cells, which are cultured in the presence of antigen/peptide for a couple of days. For the last 16-20 hours thymidine labelled with tritium (3H-thymidine) is added. Cells are then harvested onto a filter plate: medium containing free radioactivity is washed away, whereas DNA sticks to the filter. Incorporated radioactivity can be quantified by means of a beta-scintillation counter determining counts-per-minute (cpm). The usual output is given as stimulation index (S.I.), which is defined as cpm of the test sample divided through cpm of the negative control.
- As secondary endpoints T-cell immunogenicity was determined by interferon gamma ELIspot. ELIspot allows quantification of peptide-specific, functional (i.e. cytokine- secreting) T cells in biological samples like human blood. The basis of the assay is that, T cells upon stimulation with a peptide specifically recognized by the T cell receptor react by secretion of cytokines like IFN-γ. This reaction can be carried out in a 96-well plate. The filter-wells of this plate are coated with a Mab specific for IFN-γ. Consequently, each cell secreting IFN-γ leaves an IFN-γ spot, which can be visualized with a subsequent color reaction. Spots can be counted using automated plate readers. Numbers obtained are a measure for the frequency of peptide-specific, IFN-γ-secreting T cells in the sample.
- As additional secondary endpoint HLA-tetramer/FACS analysis was performed. HLA class I tetramers, soluble recombinant forms of a complex of HLA molecule and antigenic peptide, bind the antigen-specific T cell receptor used for T cell recognition. By using flow cytometry with fluorescent tetramers, antigen-specific CD8+ T lymphocytes can be reliably enumerated and characterized. The assay uses HLA-A*0201 custom-made iTag™-tetramers produced by Beckman Coulter Immunomics complexed with IC41 class I epitopes.
- Subjects were classified as responders if they showed significant T-cell responses at any of
visits 4 to 11 and had no response prior treatment. In the case of pre-existing immunity (significant T-cell response against any peptide within IC41 already prior vaccination), an increase of at least 3 times of the pre-existing value was required to classify the effect as response. - As a first important result, these clinical trials confirmed the excellent safety profile of completely synthetic peptides in general and Poly-L-Arginine in particular. Furthermore, several important lessons regarding activation of human T cells were learned: in both studies, T cell responses were assessed using [3H]-thymidine proliferation and IFN-γ ELIspot assays, and flow cytometry (FACS). These assays, which have been standardized and validated at Intercell AG's Clinical Immunology Laboratory enable reliable measurements of epitope-specific T cell responses induced by vaccination. All assays were performed in compliance with Good Laboratory Practice (GLP)/Good Clinical Practice (GCP) requirements. Standardization of the blood cell isolation procedure at the different investigational sites led to a high rate of evaluable assays. However, due to the lack of inter-laboratory standardization of T cell assays, comparison of the results of this study with published data from similar trials is difficult. Cryo-preserved blood cells were used, resulting in possible underestimation of T cell responses compared with assays that utilize fresh blood.
- In the
phase 2 study population of chronic HCV patients a slightly different picture was obtained: in general, CD4+ and CD8+ T cell responses to IC41 peptides were more frequent and more vigorous in peripheral blood samples from those patients who were immunized with peptide and Poly-L-Arginine together, than in samples from those patients who were immunized with peptide or Poly-L-Arginine alone, confirming the requirement of Poly-L-Arginine as a T cell adjuvant. Vaccine responder rates were approximately two to three-fold higher in the verum groups than in the control groups (Fig. 1 ). T cell proliferation responders were more numerous in the verum groups (30-60%) than in the control groups (0-17%). - Most importantly however, interferon-gamma ELIspot responders were observed exclusively in the verum groups (
Fig. 2 ). These results demonstrated for the first time that Poly-L-Arginine is able to induce type I responses even in the setting of chronic HCV infection in patients who could not be cured by the interferon/ribavirin standard therapy. Significant Interferon-gamma ELIspot responses were detected against both HLA-class II (recognized by helper T cells) and HLA-class I (recognized by cytotoxic T cells) peptides. Analysis of data up to and including those obtained at the second immunological check (Visit 11) performed 6 months after the last immunization revealed no important differences in immunological responder rates compared with the data obtained atVisits 1 to 10, indicating sustainability of the IC41-induced immune response. The study also disclosed that T cell immunity against the virus can be raised to a level that is not too different from the one induced in healthy vaccines. Thus, immunosuppression may not be as prevalent as anticipated in patients. It remains to be elucidated, how such T cell responses can be optimally applied to reduce disease progression or ameliorate symptoms and eventually clear the infection. - Taken together, Poly-L-Arginine represents one of the first synthetic T cell adjuvants, which has consistently - from in vitro experiments up to incurable chronically infected patients - been able to induce and augment the desired kind of immune response. Its easy manufacturability, excellent safety profile and its efficacy even in such difficult settings as chronic HCV infection, make it a promising new tool in the fight against infectious diseases and cancer.
- As shown in
Figure 1 , IC41 is able of inducing a significant proliferative response in chronic HCV patients. Interestingly, neither peptides alone nor poly-L-Arginine alone have this ability, proofing the adjuvant effect of poly-L-Arginine. - As shown in
Figure 2 , IC41 can induce interferon gamma secreting T-cells in chronic HCV patients, whereas peptides alone or poly-L-Arginine alone cannot induce any response. Importantly, both CD4+ Helper-T-cells and CD8+ cytotoxic T-cells can be induced. - Interferon gamma secretion is a hallmark of type I T-cell responses. Such responses are seen during the acute phase of infection in the subset of HCV patients, who eliminate the virus and do not proceed to chronic infection. Type I T-cell responses are also seen in patients undergoing standard therapy with interferon and ribavirin. Thus, induction of type I T-cell responses as achieved by IC41 is a primary goal of therapeutic vaccination against HCV.
- References
- Bellentani S, Miglioli L, Masutti F, Saccoccio G, Tiribelli C. Epidemiology of hepatitis C virus infection in Italy: the slowly unraveling mystery. Microbes Infect. 2000 Nov;2(14):1757-63.
Liang TJ, Rehermann B, Seeff LB, Hoofnagle JH. Pathogenesis, natural history, treatment, and prevention of hepatitis C. Ann Intern Med. 2000 Feb 15;132(4):296-305.
Cornberg M, Wedemeyer H, Manns MP. Treatment of chronic hepatitis C with PEGylated interferon and ribavirin. Curr Gastroenterol Rep. 2002 Feb;4(1):23-30.
Keilholz U, Weber J, Finke JH, Gabrilovich DI, Kast WM, Disis ML, Kirkwood JM, Scheibenbogen C, Schlom J, Maino VC, Lyerly HK, Lee PP, Storkus W, Marincola F, Worobec A, Atkins MB. Immunologic monitoring of cancer vaccine therapy: results of a workshop sponsored by the Society for Biological Therapy. J Immunother. 2002 Mar-Apr;25(2):97-138. Review.
Fried M, Shiffman M, et al. Peginterferon Alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med, Vol. 347, No. 13. September 26, 2002; 975-982.
Claims (9)
- Use of polycationic peptides for the preparation and manufacturing of medicaments or pharmaceutical compositions, comprising HCV antigens or HCV epitopes for the treatment of patients with HCV chronic infections.
- The use according to claim 1, wherein the medicaments or pharmaceutical compositions are vaccines.
- The use according to claim 1 or 2, characterised in that the patients are those who had not responded to, partially responded to or had relapsed from primary standard HCV therapy by a combination of pegylated interferon-alpha and the antiviral rib-' avirin.
- The use according to any one of the claims 1 to 3, characterised in that the polycationic peptides contain between 20 and 500 amino acid residues.
- The use according to claim 4, characterised in that the polycationic peptides contain between 30 and 200 amino acid residues.
- The use according to any one of claims 1 to 5, characterised in that the polycationic peptide is polyarginine or polylysine.
- The use according to any one of claims 1 to 6, characterised in that the medicament is used for induction of CD4+ Helper-T-cells and CD8+ cytotoxic T-cells.
- The use according to any one of claims 1 to 7, characterised in that the medicament is used for replacing or supplementing a HCV standard therapy with interferon alpha and ribavirin.
- The use according to claim 8, characterized in that the medicament is for patients where such standard therapy is not effective or not effective anymore.
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CA (1) | CA2583026A1 (en) |
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EP1648502B1 (en) * | 2003-07-11 | 2010-12-01 | Intercell AG | Hcv vaccines |
WO2006045677A1 (en) * | 2004-10-29 | 2006-05-04 | Intercell Ag | Hcv vaccines for chronic hcv patients |
CN101426514A (en) * | 2006-04-25 | 2009-05-06 | 英特塞尔股份公司 | HCV vaccines |
US8993228B2 (en) | 2008-09-30 | 2015-03-31 | Toray Industries, Inc. | Antibody binding to envelope protein 2 of hepatitis C virus and method for identifying genotype of hepatitis C virus using the same |
US8765148B2 (en) | 2010-02-19 | 2014-07-01 | Valneva Austria Gmbh | 1C31 nanoparticles |
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US5350671A (en) * | 1987-11-18 | 1994-09-27 | Chiron Corporation | HCV immunoassays employing C domain antigens |
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DE19803453A1 (en) * | 1998-01-30 | 1999-08-12 | Boehringer Ingelheim Int | vaccine |
US20090130135A1 (en) * | 1999-10-01 | 2009-05-21 | Michael Buschle | Hcv vaccines |
AT408721B (en) * | 1999-10-01 | 2002-02-25 | Cistem Biotechnologies Gmbh | PHARMACEUTICAL COMPOSITION CONTAINING AN ANTIG |
DE60130877T2 (en) * | 2000-04-14 | 2008-07-17 | Intercell Ag | MODIFIED PEPTIDE-CONTAINING PHARMACEUTICAL PREPARATIONS |
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US6337317B1 (en) * | 2000-06-27 | 2002-01-08 | The University Of British Columbia | Antimicrobial peptides and methods of use thereof |
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AT410798B (en) * | 2001-01-26 | 2003-07-25 | Cistem Biotechnologies Gmbh | METHOD FOR IDENTIFYING, ISOLATING AND PRODUCING ANTIGENS AGAINST A SPECIFIC PATHOGEN |
JP2005533855A (en) * | 2002-07-24 | 2005-11-10 | インターツェル・アクチェンゲゼルシャフト | An antigen encoded by another reading frame from a pathogenic virus. |
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